The invention relates to protein binding assays for immunosuppressant drugs in body fluids. More specifically, it relates to the isolation of a novel immunophilin from lymphatic tissues that appears to be identical to ubiquitin, and to the uses of both proteins for protein binding assays for FK-506 and rapamycin, and pharmacologically active metabolites and chemical derivatives thereof.
FK-506 (also known as tacrolimus), rapamycin (RAP, also known as sirolimus), and cyclosporin A, isolated from soil microorganisms, as well as metabolites and derivatives thereof, are currently widely used in organ transplantation to suppress the immune system, and thereby avoid organ rejection; hence, they are referred to as immunosuppressant agents or drugs.
Such drugs are candidates for precise therapeutic drug monitoring, for several reasons. One, because there are serious consequences of both undermedication (organ rejection) and overmedication (infection and toxic side effects). Two, there are wide intra- and inter-individual variations and narrow therapeutic indices. Three, the immunosuppressant drugs are known to be actively metabolized by the patients, thereby producing a mixture of pharmacologically active and pharmacologically inactive metabolites. Only approximately ⅓, xc2xe and xc2xd of CsA, FK-506 and RAP, respectively, are in the blood in the form of the parent compound; the remainder consists of metabolites of these drugs. It is obvious that analytical methods for use in therapeutic drug monitoring of these immunosuppressant drugs must (a) be able to distinguish pharmacologically active from inactive metabolite species, and (2) do so in a manner proportional to their pharmacological potency.
A wide variety of immunosuppressant drug metabolites have already been identified. Rapamycin (sirolimus) metabolites account for at least 50% of all rapamycin species in trough blood samples, so their potential for interfering in drug assays is quite high. Rapamycin is known to have at least ten metabolites (Yatscoff et al., Ther Drug Monit 17:666 (1995)). Metabolites RM1, RM2, RM3 and RM4 have been isolated from the urine of patients receiving rapamycin; only RM1 bound specifically to the 14 kDa immunophilin (21% of parent binding) and to the 52 kDa immunophilin (25% of parent binding) (Davis et al., Clin. Biochem. 29: 303 (1996)). Other known rapamycin metabolites include: 7-0-demethyl sirolimus, 41-0-demethyl sirolimus; 32,41-0-demethyl sirolimus, (C9-C23)-OH-sirolimus; and (C1-C8 or C32-C36) OH sirolimus; all but the fourth metabolite substantially binds a 5-8 kDa immunophilin and a 52 kDa immunophilin (Davis et al., Clin. Biochem. 33:31 (2000).
Certain chemical derivatives of RAP are also biologically active as immunosuppressants. For example, Rapamycin Derivative (RAD), the derivative SDZ-RAD (40-O-(2-hydroxymethyl)-rapamycin), and the rapamycin metabolite SDZ-RAD 17,18,19,20,21,22-tris-epoxide are all known to be immunosuppressants; [see, e.g., immunosuppressant for lung transplants (Serkova et al., J. Pharm. Exp. Therap. 294:323 (2000)), and kidney transplants (Schuurman et al., Transplantation 69:737 (2000)].
Tacrolimus (FK-506) metabolites comprise about 30% of the tacrolimus species in blood. Tacrolimus is metabolized into at least nine metabolites (Jusko et al., Ther Drug Monit. 17:, 596, 606 (1995)). Structures of several are 13-demethyl tacrolimus, 15-demethyl tacrolimus, and 31-demethyl tacrolimus, and their binding to the 5-8 kDa immunophilin has been studied extensively (Davis et al., Clin. Biochem. 33:1 (2000)). The principal metabolites, M-III and M-V, have no pharmacological activity in vitro; the M-II metabolite is pharmacologically active (Soldin, Clin. Biochem., 29:439 (1996)). At least one metabolite (31-demethyl tacrolimus) shows immunosuppressive activity equal to that of its parent.
Six methods have been described to date for the analysis of the aforementioned immunosuppressant drugs in patient blood: (1) HPLC; (2) high performance liquid chromatography-mass spectrometry (HPLC-MS); (3) microparticle enzyme immunoassay (MEIA); (4) ELISA; (5) p70-S6 kinase inhibitors; and, (6) an immunophilin-binding assay (IBA). For reviews of the literature comparing these four methods, see, Davis et al., Clin. Therap. 22 (Suppl. B): pp B62-70 (2000); Soldin, Therap Drug Monit 22:44 (2000). These reviews conclude that HPLC methods suffer from precision problems because of the extensive sample preparation required. HPLC-MS method are not practical for routine clinical use. Initial studies of the MEIA and ELISA have found overestimation of immunosuppressant drug concentrations, possibly because of cross-reactivity of the antibody with drug metabolites that are not pharmacologically active. Monitoring by p70 S6 kinase inhibitions is at present only theoretical, and the assay itself is not yet optimal.
The protein binding reagents preferred for IBAs for FK-506 and RAP and pharmacologically active metabolites are certain lymphatic tissue proteins referred to as immunophilins. It is widely believed that immunophilins may be the intracellular target of the immunosuppressant drugs in a process that leads to suppression of the immune system. Because immunophilins exhibit many of the properties of a physiological receptor, they have been the proteins of choice for use in IBAs. They allow the assay to measure the parent active drug or drug metabolites selectively, even in the presence of structurally similar, but pharmacologically inactive, drug metabolites. The IBA also has the potential to be automated, a valuable characteristic for the clinical laboratory.
Immunophilins of various molecular weights have been purified from the cytosolic phases of lymphatic cells. These include a 10-12 kDa protein (Siekierka et al., U.S. Pat. No. 5,109,112); a 14.6 kDa protein (Soldin, U.S. Pat. No. 5,525,523; 5,354,845); a 17.6 kDa protein (Handschumacher et al. U.S. Pat. No. 4,722,999); a 34-37 kDa protein (Soldin, U.S. Pat. No. 5,780,307); and, a 50-52 kDa protein (Soldin, U.S. Pat. No. 5,698,448).
Although IBAs appear to be the method of choice to monitor blood concentrations of immunosuppressant drugs, commercial use of these IBAs has been thwarted by the lack of adequate supplies of the immunophilins. Supply by actual isolation from lymphatic cells is cumbersome, inefficient and expensive, and may not yield a standard product. Supply by recombinant DNA means would be ideal, but this has not yet been accomplished for the above-mentioned immunophilins. Hence, it would be ideal to have a protein available that exhibits all of the desirable binding assay properties of the known immunophilins and that is commercially available in pure form, preferably as a recombinant protein. This has now been accomplished by the identification, isolation to homogeneity and partial sequencing of a novel 8.4 kDa immunophilin from extracts of lymphatic tissues that appears to be identical to ubiquitin, which is commercially available as a recombinant protein. The discovery of the novel 8.4 kDa immunophilin, the proofs of the identity of this protein to ubiquitin, and the uses of this novel ubiquitin immunophilin in IBAs are described below.
The inventor has discovered in water-soluble extracts of lymphatic tissues a heretofore unknown protein of 8.4 kDa mass with the binding specificity and binding affinity of an immunophilin specific for FK-506 and RAP, and with no significant binding to CsA.
In a second aspect of the invention, the first 23 amino acids of the 8.4 kDa protein is identical to ubiquitin, a protein heretofore known only as a participant in proteosomal proteolytic degradation of other proteins.
In another aspect of the invention, commercial preparations of ubiquitin, including recombinant human ubiquitin, are shown to exhibit immunosuppressant drug binding specificities and affinities, as well as other biochemical properties, identical to those of the isolated 8.4 kDa immunophilin.
In still another aspect of the invention, commercial ubiquitin, as well as the isolated 8.4 kDa immunophilin, are shown to exhibit high utility as a binding reagent in IBAs.
These and other aspects of the invention will become apparent by reference to the specification and appended claims.